Nicotinic receptor-evoked hippocampal norepinephrine release is highly sensitive to inhibition by isoflurane

Abstract

Background: Inhaled anaesthetics (IAs) produce multiple dose-dependent behavioural effects including amnesia, hypnosis, and immobility in response to painful stimuli that are mediated by distinct anatomical, cellular, and molecular mechanisms. Amnesia is produced at lower anaesthetic concentrations compared with hypnosis or immobility. Nicotinic acetylcholine receptors (nAChRs) modulate hippocampal neural network correlates of memory and are highly sensitive to IAs. Activation of hippocampal nAChRs stimulates the release of norepinephrine (NE), a neurotransmitter implicated in modulating hippocampal synaptic plasticity. We tested the hypothesis that IAs disrupt hippocampal synaptic mechanisms critical to memory by determining the effects of isoflurane on NE release from hippocampal nerve terminals.

Methods: Isolated nerve terminals prepared from adult male Sprague-Dawley rat hippocampus were radiolabelled with [(3)H]NE and either [(14)C]GABA or [(14)C]glutamate and superfused at 37 degrees C. Release evoked by a 2 min pulse of 100 microM nicotine or 5 microM 4-aminopyridine was evaluated in the presence or absence of isoflurane and/or selective antagonists.

Results: Nicotine-evoked NE release from rat hippocampal nerve terminals was nAChR- and Ca(2+)-dependent, involved both alpha7 and non-alpha7 subunit-containing nAChRs, and was partially dependent on voltage-gated Na(+) channel activation based on sensitivities to various antagonists. Isoflurane inhibited nicotine-evoked NE release (IC(50)=0.18 mM) more potently than depolarization-evoked NE release (IC(50)=0.27 mM, P=0.014), consistent with distinct presynaptic mechanisms of IA action.

Conclusions: Inhibition of hippocampal nAChR-dependent NE release by subanaesthetic concentrations of isoflurane supports a role in IA-induced amnesia.

Fig 1

Nicotine-evoked NE release from rat hippocampal nerve terminals. (a) Nicotine-evoked NE release (sum ΔFR) was concentration-dependent and transmitter-selective, and maximally stimulated by 100 µM nicotine (n=4). (b) Nicotine selectively evoked NE (n=61) compared with GABA (n=43) and glutamate (n=35) release, although all three transmitters were evoked by stimulation with 5 µM 4AP (n=13, 4, 17, respectively), which evoked fractional release of NE comparable with that evoked by nicotine. Statistical comparisons of sum ΔFR values [mean (sem)] were performed by one-way anova (*P<0.05; **P<0.01; ***P<0.001 vs control). (c) Nicotine-evoked NE release (n=61) required extracellular Ca²⁺ (n=8) and was nAChR-dependent as indicated by inhibition by the non-selective nAChR blocker mecamylamine (Mec, 10 µM, n=5). Release was not inhibited by the selective β2 antagonist DHβE (30 µM, n=5) and was partially inhibited by the selective α7 antagonist MLA (10 µM, n=8) or the Na⁺ channel blocker TTX (1 µM, n=12). Statistical comparison of sum ΔFR values [mean (sem)] with control was performed by one-way anova (**P<0.01; ***P<0.001).

Fig 2

Nicotinic receptor-evoked NE release from rat hippocampal nerve terminals was potently inhibited by isoflurane. (a) Isoflurane [Iso, 0.68 (0.01) mM, n=13] inhibited nicotine (100 µM)-evoked NE release (n=61), which was reversible [no effect after 12 min washout of 0.72 (0.003) mM isoflurane, Post-Iso, n=3]. Residual release in the presence of isoflurane [0.68 (0.02) mM] was not further reduced by DHβE (30 µM, n=6), whereas MLA (10 µM, n=9) with isoflurane [0.76 (0.01) mM] and TTX (1 µM, n=9) with isoflurane [0.70 (0.01) mM] additively inhibited release. The combination of TTX with MLA (n=9) did not produce greater inhibition than TTX or MLA alone. Statistical comparison of sum ΔFR values [mean (sem)] with control was performed by one-way anova (***P<0.001), and comparisons between drug vs drug combinations were performed by the Student t-test (†P<0.05; ††P<0.01; †††P<0.001). (b) Isoflurane inhibited nicotine (100 µM)-evoked NE release [IC₅₀=0.18 (0.02) mM] more potently (P=0.02) than NE release evoked by 5 µM 4AP [IC₅₀=0.27 (0.03) mM]. Shaded area highlights the clinical concentration range of isoflurane between 0.5 and 2 times the MAC (0.35 mM). Only nicotine-evoked NE was significantly inhibited by less than clinical concentrations of isoflurane (F-test comparison with 100%, **P<0.01). Significant increases in basal NE release by isoflurane alone were evident only at concentrations >0.4 mM (∼1 MAC; inset). Data are presented as mean (sem).